This invention relates to methods of assaying for 5' to 3' exonuclease activity.
Telomeric DNA of virtually all organisms is composed of short direct repeats with clustering of G residues on the strand that forms the 3' end of the chromosome (Zakian, 1995). In some protozoans, the precise structure of the very ends of the macronuclear DNA molecules is a 10-16 base overhang of the G-rich strand (Klobutcher et al., 1981; Pluta et al., 1982; Henderson and Blackburn, 1989). In Oxytricha and related ciliates, there are proteins that bind specifically to this G-rich tail (Gottschling and Zakian, 1986; Price, 1990). In vitro, these proteins protect telomeric DNA from degradation (Gottschling and Zakian, 1986). Although it is not yet known if short, G-rich overhangs are a general feature of telomeres, genetic (Wiley and Zakian, 1995) and biochemical (Cardenas et al., 1993) studies suggest that diverse organisms have terminus-limited telomere binding proteins similar to those described in Oxytricha. These data suggest that telomeres in many organisms will be found to have short, constitutive G-tails that serve as substrates for essential telomere binding proteins.
Telomeres are essential for chromosome integrity: they protect chromosome ends from random fusion events and degradation (McClintock, 1939, 1941; Sandell and Zakian, 1993) and they ensure the complete replication of the chromosome (Watson, 1972; Olovnikov, 1973). Conventional DNA polymerases require a primer and can synthesize DNA only in the 5' to 3' direction. This enzymatic machinery is expected to leave 8-12 base gaps at the 5' ends of the newly synthesized strands after removal of the last RNA primer (Newlon, 1988). A priori, this gap is expected on only half of the telomeres, the ones on which the newly synthesized strand is made by lagging strand synthesis (FIG. 1A). Telomerase is a ribonucleoprotein that uses a sequence within its RNA as template for the addition of telomeric repeats (reviewed in Greider, 1995). This enzyme can extend the short 3' overhangs left after primer removal to generate overhangs of telomeric G-strand DNA. On the other half of the telomeres, leading strand synthesis is expected to leave a blunt end (FIG. 1A). If telomere binding proteins require a short overhang of the G-rich strand for binding, the blunt end must somehow be processed to generate such a structure (Lingner et al., 1995 and see below). Since in vitro, a blunt end does not serve as a substrate for telomerase, additional, so far unknown activities must be invoked for this processing (Lingner, et al., 1995).
In Saccharomyces cerevisiae, telomeres consist of .about.300 bp of C.sub.1-3 A/TG.sub.1-3 sequences that are necessary and sufficient for all essential functions of telomeres (see for example Wellinger and Zakian, 1989; Sandell and Zakian, 1993). The Applicant showed previously that at the end of S-phase a &gt;30 base long overhang of the G-rich strand (hereafter referred to as TG.sub.1-3 tails) occurs on yeast telomeres (Wellinger et al., 1992, 1993b, c). At other points in the cell cycle, overhangs are not detected although overhangs .ltoreq.30 bases were not detectable in these studies. Short linear plasmids also acquire TG.sub.1-3 tails immediately after replication of telomeric DNA by conventional replication (Wellinger et al., 1993c). In addition, these linear plasmids form telomere--telomere associations that are dependent on the presence of the TG.sub.1-3 tails (Wellinger et al., 1993a, c).
Oligonucleotides of single stranded G-rich telomeric DNA from ciliates, vertebrates and yeast form alternative DNA structures in vitro that depend on non-canonical base pairing of the guanines (for reviews, see Sundquist, 1990, 1993). The best described structures are G-quartets, four stranded parallel or anti-parallel helices in which four guanines are in a planar alignment to each other and in which every G has two hydrogen bonds to each of two neighboring Gs (Gellert et al., 1962; Sen and Gilbert, 1988; Sundquist and Klug, 1989; Williamson et al., 1989; Kang et al., 1992; Smith and Feigon, 1992). The formation of G-quartets by intermolecular interactions is slow and requires specific cations for stabilization of the product (Sundquist and Klug, 1989; Williamson et al., 1989; Sen and Gilbert, 1990; Hardin et al., 1991; Scaria et al., 1992). In addition, a guanine may simply base pair with another guanine to form a G:G base pair (Hobza and Sandorfy, 1987; Sundquist, 1990; Gualberto et al., 1992). One possible G:G base pairing configuration has been calculated to be second only to a G:C base pair in stability (Hobza and Sandorfy, 1987). Alternatively, a single stranded G-rich DNA strand may associate with double-stranded G:C rich DNA to form a triple helix via G.multidot.G:C pairing (Beal and Dervan, 1991; Voloshin et al., 1992; Cheng and Van Dyke, 1993; Gilson et al., 1994). At least in vitro, this association requires Mg.sup.++ ions or spermine in the reaction mixture (Kohwi and Kohwi-Shigematsu, 1988; Beal and Dervan, 1991).
At least two alternatives could explain the end-to-end interactions observed with in vivo generated short linear plasmids containing TG.sub.1-3 tails (Wellinger et al., 1993c). As outlined above, the individual daughter molecules left after conventional replication and TG.sub.1-3 tail formation could have a blunt end on one telomere and a TG.sub.1-3 tail on the other (FIG. 1A). In this case, the TG.sub.1-3 tail from one end could associate with the double-stranded C.sub.1-3 A/TG.sub.1-3 repeats on the other by forming a triple helix. Alternatively, if there is a mechanism to generate a TG.sub.1-3 tail on the end replicated by leading strand synthesis, both ends of individual daughter molecules could have TG.sub.1-3 tails (FIG. 1B). These TG.sub.1-3 tails could then associate via G-quartet structures or G:G base pairing.